Molecular Cloning, Expression and Purification of Muscle Fructose-1,6-Bisphosphatase from Zaocys dhumnades: the Role of the N-Terminal Sequence in AMP Activation at Alkaline pH
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Feng-wen Zhang
Abstract
An open reading frame (ORF) of snake muscle fructose 1,6-bisphosphatase (Fru-1,6-P[2]ase) was obtained by the RTPCR method with degenerate primers, followed by RACEPCR. The cDNA of Fru-1,6- P[2]ase, encoding 340 amino acids, is highly homologous to that of mammalian species, especially human muscle, with a few exceptions. Kinetic parameters of the purified recombinant enzyme, including inhibition behavior by AMP, were identical to that of the tissue form. Replacement of the Nterminal sequence of this enzyme by the corresponding region of rat liver Fru 1,6-P[2]ase shows that the activity was fully retained in the chimeric enzyme. The inhibition constant (K) of AMP at pH 7.5, however, increases sharply from 0.85M (wildtype) to 1.2mM (chimeric enzyme). AMP binding is mainly located in the Nterminal region, and the allosteric inhibition was shown not to be merely determined by the backbone of this region. The fact that the chimeric enzyme could be activated at alkaline pH by AMP indicated that the AMP activation requires the global structure beyond the area.
Copyright © 2000 by Walter de Gruyter GmbH & Co. KG
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Articles in the same Issue
- Characterization of Regulatory Elements in the 5'-Flanking Region of the GM2 Activator Gene
- Transcriptional Activity of GLI1 Is Negatively Regulated by Protein Kinase A
- Peptide Bond Synthesis: Function of the efp Gene Product
- Molecular Cloning, Expression and Purification of Muscle Fructose-1,6-Bisphosphatase from Zaocys dhumnades: the Role of the N-Terminal Sequence in AMP Activation at Alkaline pH
- The pH-Dependent Interaction of Cinnamomin with Lipid Membranes Investigated by Fluorescence Methods
- Nitric Oxide Detection and Visualization in Biological Systems. Applications of the FNOCT Method
- Processing of V-ATPase Subunit B of Mesembryanthemum crystallinum L. Is Mediated in Vitro by a Protease and/or Reactive Oxygen Species
- The Thioredoxin Boxes of Thyroglobulin: Possible Implications for Intermolecular Disulfide Bond Formation in the Follicle Lumen
- New Bivalent Thrombin Inhibitors with N? (Methyl)Arginine at the P1-Position
- Multiple Promoters Direct the Tissue-Specific Expression of Rat Mitochondrial Glycerol-3-Phosphate Dehydrogenase
- Characterization of SNP, a Novel Tissue- and Phase-Specific Nuclear Protein Expressed during the Proliferative Phase in the Oviduct of the Lizard Podarcis sicula Raf.
- Association of Betaine-Homocysteine S-Methyltransferase with Microtubules
- Re-Evaluation of Amino Acid Sequence and Structural Consensus Rules for Cysteine-Nitric Oxide Reactivity
- Expression of Human Tissue Kallikrein in Differentiated HL-60 Cells
- Erratum
